Investigating the role of natural and engineered curli fibers in mediating interactions with the gut epithelium Abstract The importance of the microbiome in maintaining gut function has fueled the study of commensals and probiotics in an attempt to identify strains with therapeutic potential. A common strategy to develop viable therapeutics is then to use the naturally occurring microbe or identify a specific molecular agent that leads to the desired effect and make it into a drug. While this approach has seen some success, it is hindered by a lack of understanding of biological mechanisms, an inability to rationally manipulate organismal fitness in the gut environment, and inadequate delivery mechanisms. We propose an alternative approach to influencing host physiological processes focused on engineering the matrix proteins produced by bacteria during host colonization. Specifically, this proposal will determine the role of naturally occurring and engineered curli fibers in mediating inflammatory processes inside the gut. Curli fibers, which are a proteinaceous component of the E. coli biofilm, have been studied extensively in the context of pathogenic strains because of their ability to mediate adhesion to host tissues and stimulate inflammatory cytokine production. However, recent evidence suggests that the fibers may also play a protective role by increasing barrier function. Interestingly, a probiotic E. coli strain (Nissle) that is commonly used to help maintain remission in inflammatory bowel disease (IBD) patients, is also known to produce copious curli fibers in vitro. We will use genetically engineered strains of Nissle to determine to what extent curli fibers play a role in mediating inflammatory processes in the gut. Simultaneously, we will create Nissle strains that are engineered to display anti-inflammatory cytokines on their curli fibers. The efficacy of these various genetically altered Nissle strains will be measured using a combination of in vitro and in vivo model systems. Notably, we will make use of a Gut-on-a-Chip system developed by our collaborators to gain insight into the molecular mechanisms of curli fiber-epithelium interactions. This system enables the study of higher-order epithelial functions, like barrier function, villus height, and adhesion, much better than conventional transwell assays. Our proposed work with this system will also help validate it as a rapid screening technique for probiotics. In combination with established mouse models of chronic gut inflammation, these model systems will facilitate efficient identification and development of microbes with therapeutic potential against chronic gut inflammation. This is also part of a broader effort in our lab to establish that biofilm matrix proteins can be a versatile new platform for therapeutic delivery and probiotic targeting.